Project Summary/Abstract Project 3 Although donor T cells play a critical role in acute and chronic graft versus host disease (GVHD), previous studies in this Program Project specifically examined the role of donor B cells as potential contributors to GVHD after allogeneic hematopoietic cell therapy (HCT). Although donor B cells do not appear to contribute to acute GVHD these studies suggested that donor B cells play an important role in the immune pathology of chronic GVHD (cGVHD). This was also supported by studies in Project 2 where murine models demonstrated a critical role for germinal center B cells in cGVHD. The role of B cells in cGVHD was confirmed in clinical trials in Project 1 demonstrating the clinical efficacy of B cell-directed therapy with rituximab in the treatment and prevention of cGVHD. Having established that donor B cells contribute to the development of cGVHD in humans, recent studies in this project focused on a detailed analysis of B cell reconstitution after HCT and identification of factors, such as B cell activating factor (BAFF), that promote B cell activation and persistence of allo and autoreactive B cells. We also developed a high throughput approach to detect and quantify antibodies specific for a panel of HY proteins as well as methods to identify B cells secreting these antibodies in patient samples. To enable monitoring of B cell responses that contribute to cGVHD in patients who do not have gender mismatched donors and where the genetic disparities that initiate allo-reactive responses are not known, we identified a novel panel of cell surface antigens that appear to be broadly targeted by donor antibodies in patients with cGVHD. Using these panels of target antigens we will undertake a series of studies to define the potential role of antibodies as predictive biomarkers of cGVHD and response to cGVHD therapies. These antigenic targets will also be used to characterize donor B cells secreting these antibodies and the signaling pathways activated in these cells. These studies will be undertaken in patients enrolled in clinical trials designed to define the efficacy of B cell-directed therapies for prevention and treatment of cGVHD described in Project 1. These studies will complement experiments in Project 2 designed to identify specific subsets of B cells and signaling pathways that play a role in unique murine models of cGVHD. Identification of agents that inhibit B cell function or fibrogenic pathways in these murine models will help focus our analysis of patient samples on these pathways. These studies will be carried out in 3 Specific Aims. 1. Characterize the development of HY-specific antibodies following allogeneic HCT. 2. Develop a panel of surface membrane antigens (SMA) frequently targeted in patients with cGVHD. 3. Identify B cell subsets secreting allo and auto-reactive antibodies that are selectively activated in patients with cGVHD.